Spermatogonial stem cells (SSCs), male germ line stem cells, provide the foundation for spermatogenesis by their ability to both self-renew and generate daughter cells, which differentiate into spermatozoa. These attributes make SSCs the key component for sustained fertility in males and the preservation of genetic lines. Signaling for mouse SSC self-renewal is mediated by the growth factor glial cell line-derived neurotrophic factor (GDNF) likely through the c-Ret proto-oncogene receptor. Using this growth factor and a defined medium, long-term (more than 6-months) in vitro proliferation of mouse SSCs occurs, which has been confirmed by maintenance of cell surface antigenic markers and ability of cultured SSCs, when transplanted to recipients, to produce donor cell-derived spermatogenesis and progeny. Thus, a unique and powerful system is available to study the molecular mechanisms of mouse SSC self-renewal. We have recently used microarray profiling to identify GDNF-regulated genes in highly enriched populations of SSCs, providing a database of genes that may constitute the core molecular machinery regulating SSC self-renewal and survival. The same growth factor and similar molecular mechanisms are likely used by other mammalian SSCs, because stem cells from many species (e.g. rat, pig, baboon and human) have been shown to proliferate in mouse testes. In addition, important molecules identified for mouse SSC self-renewal may be helpful in understanding mechanisms used by other adult tissue stem cells. The mouse SSC culture system and stem cell transplantation technique will be used to analyze the molecular regulation of mouse SSC self- renewal. The two proposed specific aims are: 1) Investigate GDNF signaling pathways involved in SSC self- renewal, and 2) Determine the biological significance of GDNF-regulated genes in SSC self-renewal. The identification of the signals required for male germ line stem cells to grow and divide will help in understanding male infertility in humans and in establishing a system to culture or grow human SSCs outside the body. Such a culture system could be used to maintain stem cells of a male during chemotherapy, and the stem cells then returned to the male's testes to reestablish fertility after therapy.